March 2011

March 30, 2011

The first cut may be the deepest but the final cut seems to be the hardest to grasp. At least in the case of cytokinesis. Contraction of an actomyosin ring narrows the connection between the two dividing cells to form an intercellular bridge. How this bridge is resolved is still a matter of debate. Many mechanisms have been proposed for this last step of cytokinesis. For example, in some organisms, vesicle fusion at the site of constriction is thought to contribute the membrane that enables cell separation. Now, Guizetti et al. propose a new mechanism: membrane scission by ESCRT-III helical filaments. Using GFP-labeled alpha tubulin, the authors examined microtubules at the intercellular bridge in HeLa cells. They noticed that microtubules partially disassembled on one side of the bridge, on the same side as the midbody (the remnant of the central spindle that asymmetrically localizes to the constriction site). This partial disassembly occurred coincident with abscission. Using correlative live-cell and transmission electron microscopy, the authors noticed electron dense membrane ripples adjacent to the midbody (first observed in a very old JCB paper!) in the zone where the microtubules had disassembled.

Guizetti et al. show that the microtubule severing enzyme spastin is important for microtubule disassembly at the constriction site. Furthermore, ESCRT-III, which is known to localize to the intercellular bridge and is essential for abscission, was enriched at the constriction zone (whereas actin and vesicles were not). The electron-dense ripples at the constriction zone were absent in cells depleted of an ESCRT-III subunit. ESCRT-III is targeted to the intercellular bridge by CEP55, a centrosomal protein, and ALIX, an ESCRT-related protein, both of which localize to the midbody at the time of abscission. Electron tomography of the constriction zone in HeLa cells revealed cortical filaments perpendicular to the microtubule bundles. These filaments formed single or intertwined helices across the intercellular bridge and were absent in cells depleted of the ESCRT-III complex by RNAi. The authors proposed that these helical filaments are composed of ESCRT-III and that this complex provides the contractile force to deform the membrane. This is in line with ESCRT-III function in other contexts. Whether ESCRT-III is the molecular component of these helices and whether this mechanism occurs in vivo still remains to be shown. Despite these major unresolved issues, the cutting-edge microscopy and interesting results make this paper worth checking out.

In brief, two other papers caught my eye this week. In the first, Puneet et al. show that you can take a pathogenic molecule that is used to subvert the host immune system and exploit it to the potential benefit of the host. Sepsis, which is a major health threat, occurs when the human body undergoes a tremendous inflammatory reaction in response to an infection. The parasitic nematode, Acanthocheilonema viteae, produces a molecule called ES-62 that dampens the host innate immune system. ES-62 downregulates signaling by TLR4 and TLR2—pattern-recognition receptors that recognize pathogenic molecules (TLR4, for example, recognizes the bacterial outer membrane protein lipopolysaccharide). The authors show that ES-62 dampens these pathways by targeting MyD88—an adaptor of both of these pattern-recognition receptors—for degradation via the autophagosome (for more about autophagy and the immune system see review by Saitoh and Akira). Importantly, when administered to mice before or after induction of sepsis, the molecule protected the majority of mice from sepsis-induced death. The potential therapeutic benefits of these results could be quite promising.

The second paper is about how ERK/MAP kinase controls lamellipodial protrusion during cell migration. It was known previously that ERK was important for adhesion disassembly by phosphorylating the focal adhesion components FAK, Paxillin and MLCK. Now, Mendoza et al. show that ERK localizes to the leading edge and phosphorylates and activates the WAVE2 regulatory complex (WAVE2 and Abi) that activates the actin nucleation complex Arp2/3. Thus, this kinase that can respond to extracellular signals coordinates both focal adhesion disassembly and lamellipodial protrusion, two key events in cell migration.

March 21, 2011

Time for a quick roundup of highlights from today’s new issue of the JCB. Our cover image comes from Yoshimi Endo Greer and Jeffrey Rubin, who reveal how a specific isoform of casein kinase 1 (CK1) localizes to the centrosome to promote neurite outgrowth. CK1 delta promoted the growth of neurites in response to the signaling molecule Wnt-3a, whereas the closely-related isoform CK1 epsilon inhibited neurite extension. As shown on the cover, the difference lies in CK1 delta’s (green) localization to the centrosome (red). Transferring CK1 delta’s centrosome localization signal to CK1 epsilon allowed the latter isoform to promote neurite growth in place of CK1 delta. You can read more here.

Meanwhile, Albers et al. describe how the Wnt receptor Frizzled-9 (Fzd-9) stimulates bone formation. Mice lacking Fzd-9 have fragile bones, as their bone-forming osteoblasts fail to properly mineralize the extracellular matrix. Loss of Fzd-9-mediated Wnt signaling results in the down regulation of a ubiquitin-like molecule called Isg15, whose expression is also required for normal bone formation, the researchers show. The results suggest that Fzd-9 agonists could be attractive therapeutic targets for stimulating bone formation is patients with osteoporosis. You can read more about these therapeutic implications in this summary article.

Hong et al. investigate how cadherin adhesion molecules change their interactions as they enter and exit intercellular junctions. Cadherins on neighboring cells can interact with each other in two distinct ways: forming either a high-affinity strand-swapped dimer or a weaker X-dimer. Hong et al. show that cadherin molecules can directly form stable stand-swapped dimers when they enter intercellular junctions. But to leave the adhesions, cadherins break apart via an X-dimerized intermediate. In this week’s In Focus, senior author Sergey Troyanovsky likens the rearrangement in cadherin interactions to the behavior of tubulin monomers as microtubules switch between assembly and disassembly modes. Troyanovsky further explaions that the different cadherin interactions allow cells to form strong adhesive bonds rapidly, yet disassemble them just as quickly – an important property as cells must constantly remodel their adhesions throughout development. In fact, elsewhere in this issue, Buzz Baum and Marios Georgiou review how adherens junction dynamics help maintain epithelial integrity during homeostasis and tissue remodeling.

Dunsch et al. investigate how the spindle-associated protein astrin promotes chromosome alignment during metaphase. They find that astrin is targeted to the dynamic plus-ends of microtubules by a protein called kinastrin. The image to the right shows astrin tracking plus ends over the course of a series of overlaid time-lapse images (colors indicate different time points).

Laporte et al. examine how cells form cytokinesis nodes – discrete protein clusters that assemble at the cell equator before coalescing to form the contractile ring that separates daughter cells at the end of mitosis. The researchers analyzed a series of fission yeast mutants to define the order of assembly of the different node components, including myosin II, the actin-bundling IQGAP protein Rng2, the membrane-bending F-BAR protein Cdc15, and the actin-nucleating formin Cdc12. Laporte et al. then used a single molecule imaging technique called SHREC to examine how these different components are organized within each node, and found that the head domain of myosin II points into the cell interior in an orientation that may help it capture actin filaments nucleated from neighboring nodes, pulling the clusters into a mature contractile ring. Find out more by reading this summary.

Meanwhile, Bernal and Venkitaraman find that the DNA replication initiator Orc6 surprisingly contributes to the final step of cytokinesis. The authors use a neat trick to specifically deplete Orc6 at different points in the cell cycle. As expected, removing Orc6 in S phase causes problems with DNA replication, leading to G2 arrest and centrosome amplification. But depleting Orc6 during mitosis causes the abscission step of cytokinesis to fail.

Lot's more to discover on the table of contents but I'll leave you for today with this month’s biosights video podcast features Valerie Doye, from the Insitut Jacques Monod in Paris, who explains her lab’s recent discovery that the nucleoporin Nup133 anchors a network of proteins, including the motor protein dynein, that tethers centrosomes to nuclear pores in early prophase to assist the early stages of mitotic spindle assembly. You can watch the video below, or subscribe in iTunes.

March 15, 2011

It never ceases to amaze me when there are new interactions discovered at the kinetochore, given how much attention is focused on it and how well-studied many of its components are. For example, two recent papers now show that the centromeric protein CENP-C, which has been scrutinized for at least a couple of decades, connects the inner and outer regions of the kinetochore.

Screpanti et al. show in human cells that the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex (which is part of the outer kinetochore KMN network of the protein complexes Mis12-Ndc80-Knl1). Cenp-C is part of the CCAN (constitutive centromere-associated network) and is a part of centromeric chromatin. Overexpression of the N-terminus of Cenp-C prevented the localization of the KMN network to kinetochores. Predictably, these cells had chromosome segregation and spindle assembly checkpoint defects. Meanwhile, Przewloka et al. show that each protein complex of the KMN network interacts with Cenp-C in flies, which lack many of the other proteins of the CCAN. These authors targeted the N-terminus of Cenp-C to centrosomes and this recruited the other complexes of the KMN network and of course caused segregation defects. So it seems that Cenp-C is the conserved connector between the inner and outer kinetochore.

March 07, 2011

Time for a quick round-up of highlights from today's new issue of The JCB. Our cover image shows a lone Tuft cell (expressing the tracscription factor SOX9 (red) in its nucleus) sitting in the epithelium of an intestinal villus. Gerbe et al. define these cells as a new secretory cell lineage in the intestine. Characterized by a thick apical brush of microvilli, Tuft cells have been seen in various epithelial tissues, but little is known about their function due to a lack of Tuft cell-specific markers. Gerbe et al. identify a unique expression signature for intestinal Tuft cells and show that they are derived from the same stem cell population that gives rise to the four other types of intestinal epithelial cells. However, Tuft cell differentiation requires distinct transcription factors, indicating that they form a separate lineage of epithelial cells. Tuft cells produce opioids and prostaglandins, suggesting that they may control gut movements and pain, and promote both inflammation and tumorigenesis. Senior author Philippe Jay tells me here how they plan to find out more about the function of Tuft cells.

Choi et al. describe how microtubules contribute to the onset of Parkinson’s disease. The exact mechanism of dopaminergic neuron death in Parkinson’s is unknown, but many lines of evidence point to defective mitochondrial complex I as a major root cause. The drug rotenone, for example, is a complex I inhibitor that induces Parkinson’s-like symptoms in rodents. But Choi et al. demonstrate that rotenone actually kills dopaminergic neurons by disrupting their microtubules, resulting in the accumulation of cytoplasmic dopamine and the generation of reactive oxygen species that initiate cell death. You can learn more about mitochondrial complex I's potential innocence and the cytoskeleton's guilt in this week's In Focus article.

Yabuta et al. describe how a Tudor family protein called TDRD5 controls multiple stages of sperm production. TDRD5 keeps retrotransposons silent during germ cell meiosis, and promotes spermatid development by assembling RNA-processing granules required for the expression of key maturation genes. More here.

And Agarwal et al. suggest that only a small proportion of the proteins that gather round a DNA double-strand break are actively involved in repairing the damage. The researchers studied the dynamics of a mutant version of the DNA repair protein Rad54 that abolished its ATPase activity, thereby slowing the protein's dissociation from DNA. Surprisingly, this mutation immobilized only 10% of the Rad54 at each DNA damage site, indicating that most of the protein that accumulates around double-stand breaks isn't actually involved in repairing the damage by homologous recombination. You can find out more in this summary.

Elsewhere, Schmidt et al. reveal how the integrin-activating protein kindlin-3 is required for bone turnover. The picture to the right shows that, unlike wild-type cells (left), osteoclasts lacking kindlin-3 (right) fail to form “sealing zones”, structures bound by a ring of actin (green) and vinculin (red), that the cells use to attach to and resorb bone. As a result, mice lacking kindlin-3 suffer from osteopetrosis, i.e. dense bones. And Laursen et al. reveal how integrin signaling in oligodendrocytes regulates the synthesis of myelin basic protein during nerve cell myelination. You can learn more about this story in this month's edition of our biobytes podcast, in which you can also hear Judith Campisi discuss her recent review of the many different - and often conflicting - functions of cellular senescence, from tumor suppression and wound repair, to tumor promotion and aging. You can subscribe in iTunes, or listen below...

There are two great reviews in today's new issue as well, both focusing on nuclear organization. Rajapakse and Groudine discuss how the spatial arrangement of chromatin can affect gene expression, while Zimmer and Fabre describe the lessons that can be learnt from studing nuclear organization in yeast.

Lots of other interesting articles in today's new issue as well. As always, you can find them on our table of contents by clicking here.

March 02, 2011

Recently, Overholtzer et al. described a new kind of cell process called ‘entosis’ where cells engulf other cells. Distinct from phagocytosis since the engulfed cell is not dying, entosis has been observed in both cultured cancer cell lines and tumors that are unattached to the extracellular matrix. Now, Krajcovic et al. report that entosis can contribute to aneuploidy. These authors noticed that in tumor cells and cultured cells bearing these cell-in-cell structures, the host cell (which has engulfed the other cell) is frequently multinucleate. Taking a closer look, the authors imaged unattached cells in tumors and in culture undergoing division and noticed that the host cell displayed a high frequency of cytokinesis failure. This failure occurred when the engulfed cell blocked the division plane of the host cell and resulted in the formation of binucleate cells that, upon further cell divisions, become aneuploid. The authors call this a ‘non-genetic’ mechanism to generate aneuploidy since no mutation was induced (though clearly mutations may exist in the genomes of the tumor cells). Skepticism aside, entotic cells could be a good model to study the role of aneuploidy in cancer since cellular control points (like the spindle assembly checkpoint) should theoretically be functioning normally and no experimental mutations have been induced.

DeGennaro et al. identify a new gene, jafrac1, involved in germ cell adhesion in the fly Drosophila melanogaster. In this organism, germ cells are set aside early in development and migrate as a group through the developing midgut to the posterior of the embryo. Germ cells lacking jafrac1, which encodes a peroxiredoxin, show defects in adhesion to the midgut during gastrulation and as a result get excluded from the midgut. Peroxiredoxins are enzymes that act as antioxidants by degrading hydrogen peroxide. In line with this enzymatic function, jafrac1 mutants were more sensitive to oxidative stress. This group had shown previously in the JCB that dynamic regulation of DE-cadherin and cell adhesion is important for germ cell migration.

Now DeGennaro et al. show that Jafrac1 is upstream of DE-cadherin. The authors show that exposure to hydrogen peroxide causes a downregulation of DE-cadherin and beta-catenin, both components of adherens junctions. So it seems that peroxiredoxin, by reducing hydrogen peroxide levels, affects the stability of adherens junctions. I admire the fact that the authors did not simply dismiss the jafrac1 phenotype or attribute it to a housekeeping function. Although the details of this signaling pathway are still to be defined, it demonstrates that reactive oxygen species are playing very specific signaling roles during development.

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